Pseudouridine monophosphate glycosidase: a new glycosidase mechanism.
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abstract
Pseudouridine (), the most abundant modification in RNA, is synthesized in situ using synthase. Recently, a pathway for the degradation of was described [Preumont, A., Snoussi, K., Stroobant, V., Collet, J. F., and Van Schaftingen, E. (2008) J. Biol. Chem. 283, 25238-25246]. In this pathway, is first converted to 5'-monophosphate (MP) by kinase and then MP is degraded by MP glycosidase to uracil and ribose 5-phosphate. MP glycosidase is the first example of a mechanistically characterized enzyme that cleaves a C-C glycosidic bond. Here we report X-ray crystal structures of Escherichia coli MP glycosidase and a complex of the K166A mutant with MP. We also report the structures of a ring-opened ribose 5-phosphate adduct and a ring-opened ribose MP adduct. These structures provide four snapshots along the reaction coordinate. The structural studies suggested that the reaction utilizes a Lys166 adduct during catalysis. Biochemical and mass spectrometry data further confirmed the existence of a lysine adduct. We used site-directed mutagenesis combined with kinetic analysis to identify roles for specific active site residues. Together, these data suggest that MP glycosidase catalyzes the cleavage of the C-C glycosidic bond through a novel ribose ring-opening mechanism.
